Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most important infectious diseases in humans worldwide. Very little is understood regarding how Mtb communicates, despite the fact that quorum sensing (QS) normally plays an important role in virulence. QS is mediated by small molecules synthesized during secondary metabolism. In Mtb, the only signaling molecule identified is resuscitation promoting factor (Rpf), a lysozyme-like protein that can stimulate replication. We examined supernatants using QS sensors in bacterial species closely related to Mtb, focusing on Actinomycetales, particularly Streptomyces. Nearly all QS molecules have been identified using sensors, but all sensor strains are Gram negatives, making it unsurprising that previous studies have not identified QS molecules from Mtb. We reasoned that use of a Streptomyces sensor would be more likely to identify Mtb QS molecules. In our preliminary studies we identified two QS molecules, MAI-1 and MAI-2, that have the ability to induce QS pathways in Streptomyces griseus and S. coelicolor inducing sporulation and pigmented antibiotic production, respectively. Purification and analysis of the phenotypic effects of MAI-1 and MAI-2 demonstrated that they impact virulence-related phenotypes of Mtb including biofilm formation and host cell infection. Furthermore, we have shown that MAI-1 and MAI-2 regulate QS gene expression in Streptomyces by qRT-PCR and control several important global regulons involved in Mtb virulence by microarray. In the proposed small project, we plan to complete our preliminary studies to allow publication and formulation of a solid more comprehensive project to study QS in Mtb and other mycobacterial species. These experiments will be accomplished through two specific aims: 1) Complete structures of MAI-1 and MAI-2. Our working hypothesis is that the mycobacterial MAI are ringed molecules with modified aliphatic side chains similar to g-butyrolactone (GBL) QS molecules in Streptomyces. We have obtained a great deal of structural information regarding MAI-1 and MAI-2, including full H-H and H-C NMR as well as MS and IR spectral data. The two pieces of data missing are confirmation of the linkage of the aliphatic side chain to the phenolic ring and length of the side chains. In this aim, we will complete large-scale purification of MAI- 1 and MAI-2 to allow improved carbon-carbon NMR and HR-MS studies. 2) Validate MAI-1 and MAI-2 virulence networks in Mtb. Our working hypothesis is that MAI-1 and MAI-2 induce key regulatory networks that are known to be involved in virulence. Our microarray analyses found that MAI-1 and MAI-2 both induce MprA, DosR and ClgR regulons that have been shown to play a role in virulence. We will validate these data by determining the extent of regulon control by the MAI using qRT-PCR and confirm production of MAI under growth conditions that induce these regulons. Our long-term goal is to gain insight into whether MAI are involved in reactivation from latency, as suggested by our microarray studies.